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Transcriptional Profiling of Zygosaccharomyces Bailii Early

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Transcriptional Profiling of Zygosaccharomyces Bailii Early www.nature.com/scientificreports OPEN Transcriptional profling of Zygosaccharomyces bailii early response to acetic acid or copper Received: 10 May 2018 Accepted: 31 August 2018 stress mediated by ZbHaa1 Published: xx xx xxxx Miguel Antunes, Margarida Palma & Isabel Sá-Correia The non-conventional yeast species Zygosaccharomyces bailii is remarkably tolerant to acetic acid, a highly important microbial inhibitory compound in Food Industry and Biotechnology. ZbHaa1 is the functional homologue of S. cerevisiae Haa1 and a bifunctional transcription factor able to modulate Z. bailii adaptive response to acetic acid and copper stress. In this study, RNA-Seq was used to investigate genomic transcription changes in Z. bailii during early response to sublethal concentrations of acetic acid (140mM, pH 4.0) or copper (0.08 mM) and uncover the regulatory network activated by these stresses under ZbHaa1 control. Diferentially expressed genes in response to acetic acid exposure (297) are mainly related with the tricarboxylic acid cycle, protein folding and stabilization and modulation of plasma membrane composition and cell wall architecture, 17 of which, directly or indirectly, ZbHaa1-dependent. Copper stress induced the diferential expression of 190 genes mainly involved in the response to oxidative stress, 15 ZbHaa1- dependent. This study provides valuable mechanistic insights regarding Z. bailii adaptation to acetic acid or copper stress, as well as useful information on transcription regulatory networks in pre-whole genome duplication (WGD) (Z. bailii) and post-WGD (S. cerevisiae) yeast species, contributing to the understanding of transcriptional networks’ evolution in yeasts. Zygosaccharomyces bailii is described as the most problematic food spoilage yeast due to its remarkable high tolerance to weak acids, namely acetic acid1. Compared to Saccharomyces cerevisiae, Z. bailii displays a three-fold higher tolerance to this acid2. Tis marked diference has brought much interest in uncovering the molecular mechanisms underlying Z. bailii tolerance to acetic acid stress, compared with the model yeast S. cerevisiae (a topic recently reviewed by Palma et al.3). A large part of what is currently known regarding the global molecular mechanisms underlying S. cerevisiae response and tolerance to sub-lethal or lethal concentrations of acetic acid comes from the consolidation and exploitation of diverse functional genomic approaches employed throughout the last two decades3. However, the utilization of this kind of approaches in a non-conventional yeast species such as Z. bailii is still scarce, partially due to the fact that only recently the annotated genome sequences of Z. bailii strains4,5 or Z. bailii-derived hybrid strains6,7 were released. Tis genomic data, not only provided new insights into the genetic and physiological traits of Z. bailii sensu lato clade, but also rendered available fundamental genomic information for the elucidation of tolerance mechanisms to acetic acid at a genome-wide scale3. Diferent functional genomic-based approaches have been conducted to examine the biological processes involved in Z. bailii adaptation and tolerance to acetic and lactic acids, specifcally, two-dimensional gel electro- phoresis (2DE)-based expression proteomics8,9, metabolomics10, plasma membrane lipidomics11 and transcrip- tomics7. However, the genome-wide regulation of transcriptional alterations occurring in Z. bailii in response to acetic acid-induced stress is still unexplored. To date, only two transcription factors were demonstrated as being involved in Z. bailii tolerance to acetic acid, specifcally, ZbMsn412, the single homologue of S. cerevi- siae Msn4 and Msn2 general stress response activators13, and ZbHaa114, the homologue of S. cerevisiae tran- scription factor Haa1, the master regulator required for the direct or indirect activation of 80% of the acetic acid-responsive genes in S. cerevisiae15–17. Haa1 was frst identifed as a Cup2 (alias Ace1) paralogue based on sequence homology to the Cu-activated DNA binding domain and N-terminal Zn module18. However, contrarily iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal. Correspondence and requests for materials should be addressed to I.S.-C. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:14122 | DOI:10.1038/s41598-018-32266-9 1 www.nature.com/scientificreports/ to what the sequence homology to Cup2 would indicate, the function of Haa1 is not afected by the copper status of the cell18. Haa1-mediated transcriptional activation requires its interaction with the DNA binding sequence 5′-(G/C)(A/C)GG(G/C)G-3′, designated Haa1 responsive element (HRE), present in the promoter region of acetic-acid-responsive genes17. ZbHaa1 was found to be required for the adaptive response and tolerance to both acetic acid and copper stress by Z. bailii, activating the transcription of genes homologous to S. cerevisiae Haa1 and Cup2 targets, under acetic acid- or copper-induced stress, respectively14. Terefore, ZbHaa1 was proposed as a bifunctional transcription factor, assuming the functions of S. cerevisiae paralogues Haa1 and Cup2 originated afer the whole genome duplication (WGD) event14. Te aim of the present study is to examine the alterations occurring in the transcriptome profle of Z. bailii IST302 cells during early response to acetic acid- or copper- induced stress mediated by ZbHaa1. Te strain IST302 was used herein since its annotated genome was recently released, is haploid, and much more amenable to genetic manipulation and physiological studies than other studied strains5. Tis study allowed the identifca- tion of ZbHaa1-dependent regulons active during the early adaptive response to acetic acid- or copper- induced stress. It also provides useful information to allow the comparison of regulatory networks in a pre-WGD yeast species (Z. bailii) and a post-WGD species (S. cerevisiae) and to gain insights into the evolution of transcriptional networks in yeasts. Results Efect of acetic acid or copper stress in the growth of Z. bailii IST302 and derived mutant with the ZbHAA1 gene deleted. In order to evaluate the genome-wide transcriptional changes occurring dur- ing early response of Z. bailii IST302 and of the derived deletion mutant Zbhaa1∆ to acetic acid (140 mM, pH 4.0) or CuSO4 (hereafer designated as copper) (0.08 mM, pH 4.0) stress, unadapted exponentially growing cells of the two strains were inoculated under standardized conditions (Fig. 1a,b). Afer 1 hour of cultivation, acetic acid (Fig. 1c,d) or copper (Fig. 1e,f) were added to the culture medium and the cells collected afer 1 hour of exposure to the respective stress. Te efects of these stressing conditions in the growth curves of the strains under study were characterized in detail. Te latency periods for the two strains examined when exposed to acetic acid were very distinct, with the parental strain displaying a latency period of about 10 hours while the mutant Zbhaa1∆ showed a latency period of approximately 40 hours (Fig. 1c,d). During this period of adaptation, while the concentration of viable cells did not change signifcantly immediately afer acetic acid supplementation of the parental strain culture medium (Fig. 1d), the mutant Zbhaa1∆ cell population gradually lost viability until growth resumption afer 40 hours of cultivation with acetic acid (Fig. 1d). Te diferences observed in the more rapid resumption of the exponential growth under acetic acid stress in the parental strain is an indication that, as previously described for Haa1 in S. cerevisiae15, ZbHaa1 plays an essential role during the period of adaptation to this stress. Upon exposure to copper stress, in both the parental and derived mutant strains, no apparent latency phase was observed based on culture optical density (Fig. 1e), but the maximum specifc growth rate of both strains decreased. Furthermore, based on the growth curves assessed by the concentration of viable cells, a latency period was identifed for the mutant strain afer copper supplementation, characterized by the maintenance of the con- centration of viable cells (Fig. 1f). It should be noted the aggregation of cells from both strains upon copper exposure was registered by microscopic observation and this factor might have interfered with the assessment of culture optical density and colony forming units (Supplementary Fig. S1). When cultivated in either minimal or rich media, Z. bailii IST302 does not form cellular aggregates as it was previously reported5. Nevertheless, the diference in the growth curves of both strains is remarkable and an indication that ZbHaa1 also plays an essential role in adaptation and tolerance to copper stress, as observed for acetic acid-induced stress and as previously reported14. Transcriptional profiling of the early response of Z. bailii IST302 to acetic acid- or copper- induced stress. Te analysis of the changes occurring in the transcriptome of the parental strain when exposed to acetic acid or copper stress, compared with the control condition, led to the identifcation of the dif- ferently expressed genes (DEGs), using as cut-of values a Fold change > 1.50 and an FDR < 0.05. Te identifed genes were submitted to a Gene Ontology (GO) term enrichment analysis by running a Fisher Exact Test with Blast2GO19. For the concentration of acetic acid tested (140 mM at pH 4.0), 297 genes were found to exhibit signifcant changes in the transcription
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